Cardiotropic formulation
The invention provides a cardiotropic formulation comprising a cardiac glycoside, a beta-adrenergic receptor stimulant nicotinamide adenine dinucleotide, cytochrome C and inosine for use in conditions of heart failure.
The present invention relates to cardiology, and more particularly it relates to the preparation of cardiotropic medicaments having a potent effect in severe and refractory conditions of cardiac insufficiency.
BACKGROUND ARTKnown in the are is a number of cardiotropic medicaments, in particular a pharmaceutical composition, Ildamen-novodigal (patent No. 3332 Arzneim . U.el,Forsch (Drug Res.),25,Nr.3 (1975), F. Stroman und R. Hempel. Kombination yon Oxyfedrin mit 3-acetildigoxin), which is referenced here as the prior art to a cardiotropic formulation of the invention known under trade name Refrakterin.
The Ildamen-novodigal formulation including 0.2 mg of beta-acetyldigoxin and 18 mg of oxyfedrin is in common use for treatment of a cardiac insufficiency including conditions associated with coronary circulatory failure. Depending on the severity of patient's condition a daily medication of the combination may be increased up to 0.6 mg of beta-acetyldigoxin and 36 mg of oxyfedrin, and the treatment period may be longed for 1 month. The action of the combination of beta-acetyldigoxin and oxyfedrin is based on a positive inotropic synergism. Such combination of beta-acetyldigoxin and oxyfedrin provides a considerable decrease in toxicity of the former, prevents the occurence of bradycardia and arrhythmia thereby increasing the patient's drug tolerance. However, despite wider range over curable and toxic doses of beta-acetyldigoxin combined with oxyfedrin, the gap between them is still small.
The usage of such combination may cause of intoxication, especially in cases of cardiac hypersensivity to glycosides, in patients with myocardial inflammations, particularly in patients with influenza-related or acute isolated myocarditis, and alcohol-drinker's cardiomyopathy.
The usage of beta-acetyldioxin in combination with oxyfedrin at a considerably lower dose than that of the ildamen-novodigal formulation in cardiomyocyte energy-deficient conditions, such as a well-defined heart insufficiency arising from a toxic-allergic myocarditis, may cause non-responsiveness of the myocardial contractile protein system (Tabl.2) which will aggravate the energy-deficient state of cardiac muscle cell (Tab.3).
If such compounds are used individually, the enhancement of myodynamia of contractile proteins are accompanied with a considerable rise in ATP, and yet CP (creative phosphate) decreases in content. (Tabl.3). As a consequence of the deterioration of a metabolic state, there occurs the activation of myocardial and cardiomyocyte degeneration (N. N. Kipshinidze et al., 1983, Materialy nauchnoi sessii NIl klinicheskoi i eksperimentalnoi terapii Minzdrava Gruzii). This phenomenon may be considered to be the basis of cardiac refractory reaction to the conventional methods of treatment.
The object of the present invention is to provide a cardiotropic formulation, which medicative effect together with a low toxicity, will allow one to restore myocardial contractile force and increase the compensation abilities in a well-defined and severe heart failure including cardiac insufficiency with refractory reaction to the conventional methods of treatment and to cut the time for attaining positive results.
DISCLOSURE OF THE INVENTIONThe object of the invention is attained by providing a formulation, hereinafter refered to as Refrakterin, distinguished from ildamen-novodigal in that it further includes nicotinamide adenine dinucleotide (NAD), cytochrome C and inosine (riboxin), with oxyfedrin and beta-acetyldigoxin presenting in smaller amounts than that of ildamen-novodigal, at the following weight ratio of ingredients:
______________________________________
cardiac glycoside 0.05-0.2
B-adrenoceptor stimulant
0.3-3.5
nicotinamide adenine dinucleotide
0.5-5
cytochrome C 5-15
inosine (riboxin) 20-250
______________________________________
From the results of our fundamentals follows that severe cardiac insufficiency in various heart diseases at the stage of too far progression of it and in conditions with refractory reaction occuring at myocardial inflammations, alcohol-drinker's cardiomyopathy (and probably at other cardiac diseases) substantially is associated with a simultaneous disturbance of three subcellular systems which are responsible for cardiomyocyte contraction-relaxation including the contractile proteins system, transmembrame Ca transport (coupling the excitation--contraction, regulating the power of contraction and evoking the relaxation of cardiac muscle) and the metabolizable energy system (ensuring an easily available energy source for utilization of it by all cellular systems) (Tabl.1).
Thus, to overcome the problems associated with a refractory reaction and provide a successful recovery of the power failure of the heart a homeostatic state for normal function of the three systems as mentioned above is required, since the activation of one (or two as it is the case of ildamen-novodigal) of such three systems does not cause the normalization of cardiac contractile function, and there occur refractory reaction to the preparation used. In addition, cardiomyocyte structural and functional abnormalities may occur because of the increased (non-compensable) ATP and the aggravation of energy depletion. It is precisely the picture as indicated above which is under observation in myocardial inflammations under ildamen-novodigal treatment, using even much less amounts than that of the original formulation (Tabl. 3).
Thus, the problem directed to overcoming the refractory reaction and attaining a fast, successful reparation of functional activities of the three cardiomyocyte systems is to restore a steady, equilibrium state of functioning of all the above cardiomyocyte systems which are responsible for myocardial contraction-relaxation. In this case, the adequate repair of metabolic activity (in sense of energetics) is of crucial importance, since the activation of function, both the contractile protein system and ionic pumps, give rise to a considerable increase in consumption of energy by cells. Most likely, it is the energy-deficient state which is the basis of the refractory reaction of severe heart failures to the conventional methods of treatment, wherein would take place a one-sided activation of the contractile protein system without the required stimulation of cellular energetic system.
The energy-deficient conditions in any heart disease accompanied with the development of myocardial hypoxia (myocardial infarction, inflammatory and alcoholic cardiac failures, terminal heart insufficiency and the like) are associated not only with the decrease in rate of cellular oxygen transport, and also with considerable losses of cellular cytochrome C, NAD and adenylate nucleotides.
The complex formulation including NAD, cytochrome C, inosine, a cardiac glycoside (.beta.-acetyldigoxin or .beta.-methyldigoxin, or strophanthin K, a contractile protein activator) and a .beta.-adrenoceptor stimulant (oxifedrin or nonachlazin, a cardiotropic sympathomimetic) has overcome the problems associated with a refractory reaction in heart insufficiency and a fast normalization of myocardial structural and functional status.
The usage of the formulation of the invention in toxic-allergic myocarditis with a well-defined power failure of the heart provides the complete restoration (Tabl.4) of normal intracardiac or peripheral hemodynamics even under cardiac hypertension (Tabl 5) This effect is accomplished by a steady equilibrium of functioning of the three cardiomyocyte systems which are responsible for contraction-relaxation reaction. In this case, the following picture develops:
The exogeneous NAD compensates the deficiency of cytosolic and mitochondrial NAD pools. This causes the normalization of NAD-dependent glycolate dehydrogenase activities, glycolytic oxidoreduction and, in turn, ATP production through the glycolytic pathway. In addition, it restores the rate of proton translocation from cytosole to mitochondria (via the malate-aspartate shuttle) for further utilization of it in the electron transport chain.
The replacement of mitochondrial NAD deficiency activates the Krebs cycle NAD-dependent dehydrogenases and regenerates proton translocation from the tricarboxylic acid cycle to the electron transport chain.
In turn, mitochondrial exogenous cytochrome C is incorporated into (on is own place) the electron transport chain. This provides the regeneration (by volume) of electron transport to oxygen and the normal mitochondrial ATP-producing capacity in the oxidative phosphorylation associated with passing the electrons through their transport chain.
The picture described may be fully developed with the proviso that the replacements of NAD and cytochrome C take place at a time.
The replacement of NAD depletion only (by capacity) does not provide the complete restoration of energetic potential of the system since the carrying capacity of the electron transport chain is still low, and the chain is unable to cope with proton flows coming from the Krebs cycle and cytosolic component. By virture of the action of exogenous NAD the level of CP does not increase to the normal (Tabl.6), whereas there is the tendency for a increase in ATP and a decrease in ATP/ADP ratio (Tabl.6,7).
The replacement of cytochrome C losses solely (which is in common use today) also does not provide the complete restoration of the ATP-producing capacity of the energy transfer system (Tabl.6), as the replenishment of cytochrome C losses will not cause the normalization of [NAD]/[NAD.H] ratio (Tabl.8 ) which regulates dehydrogenase activity. In this case, the ADP level goes low, and [ATP]/[ADP] ratio rises above-normal.
The replacement of losses, either NAD or cytochrome C (Tabl.8), does not cause the normalization of the total adenyl nucleotides (Tabl.6, 7). Their total content is still low.
The replacement of the total adenyl nucleotides may be attained by including inosine in the formulation, which promotes de novo adenyl nucleotide synthesis (Tabl. 9).
In addition, inosine enhances considerably the coronary circulation (oxygen delivery to cardiac muscle), and assists in liberating oxygen from oxyhemoglobin of peripheral blood. This, without any doubt, contributes significantly to the restoration of cardiomyocyte ATP-producing capacity that is a matter of great concern at the increasing heart stresses.
Thus, the introduction of exogenous NADs, cytochrome C and inosine into the composition for the replacement of endogenous NAD and cytochrome C losses, and apparently of inosine, makes it possible to restore a homeostatic state of the energy transfer system (normal levels of ATP, ADP, AMP, CP, NAD and NAD.H), its functional capacity,[ATP]/[ADP] and ##EQU1## as along with the absolute content of ATP, ADP, AMP, CP, NAD and NAD.H their ratios are of great importance in regulation of energetic state of the system, that is the case in hand is not a simple summation over the positive effects (each of the agents used activates ATP production to one or another degree), and it means a fine adjustment of such system. It should be noted that normal ATP and ADT are the matter of great concern for the desired structural status of contractile proteins.
In the treatment of cardiac insufficiency after restoration of normal function of the energy exchange system the next problem is to return the contraction ability of the contractile protein system (Tabl.10). This problem is solved by including a cardiac glycoside (Tabl.10) in the formulation for its direct effects on contractile proteins. The basic preparation, Refrakterin, includes .beta.-acetyldigoxin as the cardiac glycoside.
However, in cases of heart hypersensitivity to cardiac glycosides (for example in allergic heart failures, acute isolated myocarditis) a-methyldigoxin (Refrakterin, BM) would be appropriate for use, since the specific activity of .beta.-methyldigoxin is high as that of .beta.-acetyldigoxin by an order of two, with their optimum concentrations being of 10.sup.-8 and 10.sup.-5 M, correspondingly (FIG. 1, Tabl.11).
In case of acute heart insufficiency associated with energy- deficient condition, strophanthin K would be suitable for use (Refrakterin, SK) as the alternative of .beta.-acetyldigoxin because, if chosen at the optimum amount, it acts on energy transformation, both quantitatively (like .beta.-acetyldigoxin) and qualitatively (Tabl.11,14), that is it increases the contraction efficiency. In addition, the range of effective doses for strophanthin K goes far beyond that of .beta.-acetyldigoxin plus .beta.-methyldigoxin. This allows such glycoside to be used effectively in the decreased amounts than that of Refrakterin (FIG. 1).
As .beta.-methyldigoxin chosen at the optimum concentration has effects, like strophanthin K, on contractile protein-transformed energy, both quantitatively and qualitatively, Refrakterin BM is suitable in acute or chronic heart insufficiency which is followed by oxygen- and energy deficiencies.
Finally, the next problem is to provide the normal function of the Ca transport system (Tabl.13). As cardiac glycosides belonging to the foxglove class have no effects directly on the contractile protein system (strophanthin K acts on both systems) the basic formulation (Refrakterin) includes a cardiotropic .beta.-adrenergic receptor stimulant, oxyfedrin, to normalize the function of Ca transport system. Oxyfedrin promotes the intake and release of Ca from sarcoplasmatic reticulum and brings the rate of such processes to the normal values (Tabl.14). In addition, oxyfedrin has a direct effect on the contractile protein system and accelerates glycolysis of phosphorylase due to transformation of it to its active form.
Oxyfedrin has these effects through the activation of adenylate cyclase (Tabl.14a), the inhibition of phosphodiesterase activity (Tabl.14b) and the increase in content of myocardial cAMP.
Moreover, oxyfedrin has effects on the energy-producing system by increasing the ATP level, whereas there occur a considerable decrease in CP (Tabl.3).
Alternatively, oxyfedrin may be replaced with its analogue, nonachlazin (Refrakterin H), with the dose of the latter should be at least 5 times as high as that of the former (Tabl.14 c).
Thus, each ingredient of refrakterin assists in the increase in level of ATP (up to its normal), contraction ability of the contractile protein system and rate of the cardiomyocyte transmembrane Ca transport to a greater or lesser extent. However, the end result of Refrakterin is not the sum of its specific effects, and it appears to be the case of a complex regulation of functional activities of the three cardiomyocyte systems responsible for contraction-relaxation reaction to provide the complete normalization of cardiomyocyte contractile function (even under hypertension) (Tabl.5).
The formulation of the invention is prepared as follows:
NAD (0.5 mg), cytochrome C (10 mg), inosine (80 mg), oxyfedrin (0.3 mg) and beta-acetyldigoxin (0.075) (based on kg of animal body weight) are mixed together and the resultant mixture before its direct usage, is dissolved under agitating in 6 ml of a commercially available normal saline which was heated to a temperature of 38.degree.-40.degree. C. The preparation was stored at 4.degree. C. prior to dilution of it.
The present invention will become more fully apparent from the following Examples of testing the claimed preparation, Refrakterin.
EXAMPLE 1 Medicative effect of Refrakterin in a 3-day TAMToxic and allergic myocarditis was induced in rabbits according to the methods of S. V. Andreev and M. V. Sokolov ("Sanogenesis, Meditsina, 1968) by injecting twice intravenously 2 ml of horse blood serum every forth day. Seven days after completion of the last injection 0.5 ml of staphyloccocia toxin were infused.
A medical effect of Refrakterin was recorded after administration of it within a 2-day period at a daily dose of 90.5 mg/g, beginning on the second day from the disease progression.
With a 3-day TAM the administration of Refrakterin within a 2-day period returns the normal status of a peripherial and intracardiac hemodynamics including the normalization of cardiac systolic and diastolic functions (Tabl.4) and their coordination, and the liquidation of blood congestion in lungs and liver. As this takes place, cardiomiocyte functional status will be restored so firmly that heart is capable to cope even with overloads (Tabl.5)
Example II Medicative effect of Refrakterin in a 10-day TAMTAM was induced according to-the same procedure as disclosed for a 3-day TAM. A pharmaceutical activity of Refrakterin was recorded after administration of the preparation at a dosage of 90.875 mg/kg within a 5-day period. In these tests for an additional effect on the inflammation process the animals were treated by injection of Refrakterin to them followed by a simultaneous oral administration of acetylsalicylic acid in a dosage of 0.25 mg/kg body weight, beginning on the fifth day from the disease progression. In special experiments aspirin in such dosage has been discovered to have no marked effects on functional activity of any of the three cardiomyocyte systems which are responsible for the contraction-relaxation reaction.
The administration of Refrakterin every day within a five- day treatment period leads to the elimination of peripheral congestion (interstitial fluid in liver, lungs and heart itself decreases to the normal in content), the normalization of cardiac systolic and diastolic functions and their coordination, ensuring normal cardiac work not only at rest (Tabl.4), but under cardiac hypertension as well (Tabl.5)
The Refrakterin treatment of a 10-day TAM results in the coordinated repair of functional activities of the contractile proteins (Tabl.10) and cardiomyocyte transmembrane Ca transport (Tabl.13) with adequate promotion the rate of ATP production as an easily available energy source and energy accumulation as CP (Tabl.15), thereby causing a sharp improvement in cardiomyocyte ultrastructure. In this case it is paticularly remarkable that the restoration of the rate of contraction process and Ca transport with the metabolisable energy support takes place also under cardiac hypertension. Thus, the Refrakterin treatment provides a steady, equilibrium repair of functioning the three cardiomyocytes systems which are responsible for the contraction-relaxation reaction.
Comparative study of effects of Refrakterin and.beta.-acetyldigoxin-oxyferdin combination
The administration of beta-acetydigoxin in combination with oxyferdin even in considerably decreased amounts than that of ildamen-novodigal (0.15 mg/kg of beta-acetydigoxin+0.6 mg/kg of oxyferdin) for 2 days in both a 3-day- and 10-day TAM causes a sharp increase in myocardial tension which generates the contractile protein system (Tabl.16), with the level of adenyl nucleotides being the same as seen in Control. At the same time the CP content remains at Control level. The administration of such combination in much less amounts (0.05 mg/kg of beta-acetydigoxin +0.2 mg/kg oxyferdin) in allergic myocarditis and adrenaline-caffeine-related heart failure evokes the decrease not only in level of CP, but in ATP as well, due to rising the tension generated by the contractile proteins.
Thus, even a short-term administration of ildamen-novodigal in myocardial inflammations leads to the aggravation of cardiomyocyte energy-deficient state that may cause, eventually, the deterioration of myocardial structure due to the progression of parenchymatous degradation.
Industrial applicabilityA cardiotropic formulation of the invention may be used in treatment of severe and refractory conditions of cardiac insufficiency for recovery of the power force of the heart and improvement of myocardial compensation abilities as well as the cutting of time for attaining positive pharmacologic effect.
TABLE 1
__________________________________________________________________________
State in Systems of Contractile Proteins, Energy Supply
and Ca.sup.2+ transport in a 10-day TAM
Mechanical parameters of contraction
Ventricle Parameter Normal (n = 16)
TAM (n = 13)
__________________________________________________________________________
Left Tension, mH/mm.sup.2
2.48 .+-. 0.18 1.57 .+-. 0.13***
Work, nJ/mm.sup.3
21.4 .+-. 3.0 9.0 .+-. 2.0**
Right Tension, mH/mm.sup.2
2.44 .+-. 0.20 1.55 .+-. 0.15**
Work, nJ/mm.sup.3
19.9 .+-. 3.0 8.3 .+-. 2.0*
__________________________________________________________________________
Levels of Adenyl nucleotides and creatine phosphate (CP)
Energetic
Ventricle
Group
ATP ADP ATP + ADP + AMP
ATP/ADP
potential
CP
__________________________________________________________________________
Left Normal
2.50 .+-. 0.13**
2.02 .+-. 0.12*
5.59 .+-. 0.18***
1.35 .+-. 0.12
0.65 .+-. 0.02
3.40 .+-. 0.18***
TAM 1.30 .+-. 0.13
1.49 .+-. 0.15
3.56 .+-. 0.21
1.05 .+-. 0.12
0.57 .+-. 0.02
1.52 .+-. 0.07
Right
Normal
2.40 .+-. 0.13***
1.84 .+-. 0.10
5.25 .+-. 0.22***
1.43 .+-. 0.13
0.65 .+-. 0.01
3.26 .+-. 0.11
TAM 1.67 .+-. 0.12
1.51 .+-. 0.18
3.77 .+-. 0.34
1.27 .+-. 0.10
0.60 .+-. 0.19
1.45 .+-. 0.10
__________________________________________________________________________
.sup.45 Ca.sup.2+ Binding, Intake and Release from CPF, Mitochondrial Ca
Uptake and
Ca Content in CPF, mitochodria and cardiac muscle in TAM (nmole .sup.45
Ca.sup.+2 per g protein)
CPF
Ca Release
binding, Ca uptake, min. Ca Ca
Test 1 min.
3 5 10 15 25 35 45 uptake
released
__________________________________________________________________________
Normal
43.5 .+-.
834 .+-.
945 .+-.
1171 .+-.
1297 .+-.
1356 .+-.
1426 .+-.
1414 .+-.
1345 .+-.
276 .+-.
(n = 15)
199***
50***
37***
43***
27***
22***
23***
19***
29***
21**
10-day
29.4 .+-.
563 .+-.
608 .+-.
698 .+-.
780 .+-.
829 .+-.
905 .+-.
908 .+-.
938 .+-.
155 .+-.
TAM 0.9 25 128 38 39 15 36 23 35 28
(n = 7)
__________________________________________________________________________
Cardiac
CPF Mitochondria
muscle
Mytochondria content
Uptake, min. Ca per g Ca per g
Test 5 15 30 mcM protein tissue
__________________________________________________________________________
Normal
116 .+-. 9.0*
188 .+-. 5.7**
210 .+-. 7.0
49.0 .+-. 4.0***
50.0 .+-. 7.5
2.5 .+-. 0.5*
(n = 15)
10-day
159 .+-. 19
258 .+-. 23
240 .+-. 8
28.2 .+-. 1.2
33.0 .+-. 3.5
1.12 .+-. 0.2
TAM
(n = 7)
__________________________________________________________________________
TABLE 2
______________________________________
Effects of oxyfedrin (0.6 mg/kg), beta-acetyldigoxin (0.15
mg/kg) and combinations thereof on left/right ventricle tension
in a 10-day TAM (mH/mm.sup.2)
Group, Vetricle
Subgroup Left Right
______________________________________
Normal 1 2.1 .+-. 0.11
1.98 .+-. 0.14.sup.x
(n = 27)
10-day
TAM
Control (n = 9) 2 0.95 .+-. 0.15
0.88 .+-. 0.14**
TREATMENT
Oxyfedrin 3 4.2 .+-. 0.49
3.90 .+-. 0.42
(n = 5)
b-Acetyldigoxin 4 4.3 .+-. 0.48
3.8 .+-. 0.19
(n = 3)
Oxyfedrin + 5 4.93 .+-. 0.4
4.7 .+-. 0.37
b-Acetyldigoxin
(n = 3)
Least significant difference
1-2 <0.001 <0.001
between groups (P)
1-3 <0.001 <0.001
1-4 <0.001 <0.001
1-5 <0.001 <0.001
2-3 <0.001 <0.001
2-4 <0.001 <0.001
2-5 <0.001 <0.001
3-4 -- --
3-5 -- --
4-5 -- --
______________________________________
.sup.x n = 24
**n = 8
TABLE 3
______________________________________
Levels of adenyl nucleotides and creatine phosphate in
allergic myocarditis under effect oxyfedrin (0.6 mg/kg)
b-acetyldigoxin (0.15 mg/kg) and combination thereof
(.mu.M per g wet tissue)
Group ATP ADP AMP CP
______________________________________
Normal 2.38 .+-. 0.14
1.92 .+-. 0.46
0.92 .+-. 0.11
1.08 .+-. 0.08.sup.+
n = 8
Control
1.67 .+-. 0.15*
1.80 .+-. 0.14
0.84 .+-. 0.37
0.59 .+-. 0.09*
10-day
TAM
n = 10
Oxy- 2.28 .+-. 0.39
2.39 .+-. 0.27
1.73 .+-. 0.11
0.29 .+-. 0.01*.sup.x
fedrin*
n = 7
b-Acetyl-
2.28 .+-. 0.15.sup.x
2.95 .+-. 0.56
1.24 .+-. 0.31
0.41 .+-. 0.11*
digoxin
n = 5
Oxy- 1.63 .+-. 0.37
2.30 .+-. 0.61
1.15 .+-. 0.34
0.22 .+-. 0.04*.sup.x
fedrin +
b-acetyl-
digoxin
n = 4
______________________________________
Note:
.sup.+ n = 12, comparison to: normal*, Control.sup.x
*oxyfedrin
1propanone-[3(2-hydroxy-1-methyl-2-phenyl-ethyl)amino1-(3-methoxyphenyl)
TABLE 4
__________________________________________________________________________
Intracardiac hemodynamics in TAM after Refrakterin treatment
CEI by
dp/dt dp/dt Veragood
dp/dt CWI,
Heart rate,
LVP, max EDP, min CI, max mmHg .times. b/s
Group b/min.
mmHg mmHg/s
mmHg/s
mmHg/s s.sup.-1
mmHg/s .times. g
g
__________________________________________________________________________
Normal 228 .+-. 15
71 .+-. 9
1240 .+-. 306
7 .+-. 2
1313 .+-. 281
21.1 .+-. 5.0
547 .+-. 93
6679 .+-. 1365
(n = 5)
TAM
CONTROL
3-day 240 .+-. 29
81 .+-. 9
1006 .+-. 122
15 .+-. 4
720 .+-. 95*
15.8 .+-. 3.9
439 .+-. 65
4685 .+-. 203*
n = 3
10-day 202 .+-. 14
49 .+-. 8*.sup.x
1017 .+-. 174
27 .+-. 8*.sup.x
533 .+-. 105*
15.4 .+-. 1.8
409 .+-. 58*
3828 .+-. 989.sup.x+
n = 4
Refrakterin
treatment
2 day 218 .+-. 15
84 .+-. 10.sup.x+
1300 .+-. 210
8 .+-. 2.sup.x *
1180 .+-. 115
20.8 .+-. 1.6
583 .+-. 45.sup.x+
6038 .+-. 979.sup.x+
n = 4
5 day 244 .+-. 8
69 .+-. 10.sup.x+
1280 .+-. 366
5 .+-. 2.sup.x+
1033 .+-. 28.sup.x *
21.5 .+-. 5.4
637 .+-. 186.sup.+
7378 .+-. 1020.sup.x+
n = 7
__________________________________________________________________________
Note:
Comparison to: normal*; 3day TAM.sup.x ; 10day TAM.sup.
*Treatment time: 2 day in 3day TAM, 5 day in 10day TAM
TABLE 5
__________________________________________________________________________
Intracardiac hemodynamics in TAM after Refrakterin treatment
under hypertension
CEI dp/dt
dp/dt,
Veragood
by max
Heart rate,
EDP, LVP, max CI, dp/dt CWI, mmHg/s
Group b/min.
mmHg/ss
mmHg mmHg/s
s.sup.-1
mmHg s .times. g
mmHg .times. b/s
max
__________________________________________________________________________
Normal 192 .+-. 15
12 .+-. 26
112 .+-. 23
1264 .+-. 504
18 .+-. 3
558 .+-. 85
7010 .+-. 966
953 .+-. 193
n = 5
TAM
CONTROL
3-day 210 .+-. 14
27 .+-. 8*
133 .+-. 14
1650 .+-. 27
16 .+-. 4
702 .+-. 92
8277 .+-. 404
1289 .+-. 163*
n = 3
10-day 218 .+-. 19
31 .+-. 2*
141 .+-. 20
1595 .+-. 10
13 .+-. 2*
858 .+-. 255
8333 .+-. 2621
767 .+-. 436*
n = 4
Refrakterin
treatment
2 day 200 .+-. 25
16 .+-. 4.sup.x+
111 .+-. 13
1358 .+-. 110
15 .+-. 3
1360 .+-. 380
8010 .+-. 1050
980 .+-. 85.sup.x
n = 4
5 day 193 .+-. 23
18 .+-. 4.sup.+
109 .+-. 15.sup.+
1452 .+-. 269
14 .+-. 2
1766 .+-. 1103
8230 .+-. 1820
1004 .+-. 177.sup.+
n = 7
__________________________________________________________________________
Note:
Comparison to: normal*; 3day TAM.sup.x ; 10day TAM.sup.
*Treatment time: 2 day in 3day TAM, 5 day in 10day TAM
TABLE 6
__________________________________________________________________________
Levels of adenyl nucleotides and CP (mcM/g wet tissue) in myocardial left
ventricle
in rabbits with a 10-day allergic myocarditis after treatment with NAD
and cytochrome C
ATP + Energy
Group ATP ADP AMP ADP + AMP
ATP/ADP potential
CP
__________________________________________________________________________
Norm (n = 25)
2.5 .+-.
2.02 .+-.
1.07 .+-. 0.07
5.52 .+-. 0.18
1.35 .+-. 0.07
0.65 .+-. 0.02
3.40 .+-. 0.18
0.13 0.12
MYOCARDITIS
Control 1.3 .+-.
1.49 .+-.
0.78 .+-. 0.09***
3.56 .+-. 0.21***
1.05 .+-. 0.12*
0.57 .+-. 0.01***
1.52 .+-. 0.07***
(n = 20) 0.13***
0.16**
TREATMENT
NAD 2.43 .+-.
2.68 .+-.
0.58 .+-. 0.14**
5.57 .+-. 0.51.sup.++
1.14 .+-. 0.22
0.65 .+-. 0.04.sup.+
1.50 .+-. 0.12***
0.05 mg/kg
0.22.sup.+++
0.33**.sup.++
(n = 12)
Cytochrome C
2.19 .+-.
1.42 .+-.
0.62 .+-. 0.09***
4.24 .+-. 0.16***.sup.+
1.56 .+-. 0.09.sup.++
0.69 .+-. 0.02.sup.+++
2.07
.+-. 0.22***.sup.+x
5
5 mg/kg 0.09.sup.+++
0.05***.sup.xx
(n = 9)
__________________________________________________________________________
Note:
the same symbols as given in Table 2 are used
TABLE 7
__________________________________________________________________________
Effects of medication of NAD (0.05 mg/kg) and cytochrome C (10/kg) on
myocardial right-ventricular
adenyl nucleotides in rabbits with a 3-day allergic myocarditis (mcM/g
wet tissue)
Statictical Total Energetic
Group, Subgroup
values
ATP ADP AMP ATP/ADP
Nucleotides
Potential
__________________________________________________________________________
Normal (n = 25)
1 X .+-. m.sub.x
2.40 .+-. 0.13
1.84 .+-. 0.10
0.99 .+-. 0.07
1.43 .+-. 0.13
5.25 .+-. 0.22
0.65 .+-. 0.01
MYOCARDITIS
Myocarditis
2 X .+-. m.sub.x
1.55 .+-. 0.10
1.45 .+-. 0.19
0.83 .+-. 0.13
1.27 .+-. 0.17
3.88 .+-. 0.33
0.60 .+-. 0.02
(n = 11)
TREATMENT
NAD (n = 11)
3 X .+-. m.sub.x
2.06 .+-. 0.12
1.99 .+-. 0.13
0.60 .+-. 0.03
1.07 .+-. 0.11
4.73 .+-. 0.42
0.63 .+-. 0.02
Cytochrome C
4 X .+-. m.sub.x
2.00 .+-. 0.28
1.09 .+-. 0.15
0.63 .+-. 0.17
2.14 .+-. 0.28
3.82 .+-. 0.42
0.66 .+-. 0.05
(n = 8)
P.sub.1-2
0.001 -- -- -- 0.001 0.05
P.sub.1-3
-- -- 0.001 -- -- --
P.sub.1-4
-- -- 0.05 -- -- --
P.sub.2-3
0.01 0.05 -- -- -- --
P.sub.2-4
-- -- -- 0.05 -- --
P.sub.3-4
-- 0.01 -- 0.01 -- --
__________________________________________________________________________
TABLE 8
__________________________________________________________________________
Levels of NAD and NAD-H in myocardial left ventricle in a 10-day TAM
after treatment with NAD (0.5 mg/kg) and cytochrome C (5 mg/kg) (mcM/g of
wet tissue)
Groups,
Subgroups NAD NAD-H Total NAD-H/NAD
__________________________________________________________________________
NORMAL (n = 8)
0.68 .+-. 0.02
0.36 .+-. 0.04
1.08 .+-. 0.03
0.50 .+-. 0.036
TAM
Control 0.47 .+-. 0.05***
0.41 .+-. 0.05
0.84 .+-. 0.03***
0.86 .+-. 0.04***
(n = 8)
TREATMENT
NAD 0.68 .+-. 0.04.sup.+++
0.33 .+-. 0.015
1.03 .+-. 0.03.sup.+++
0.46 .+-. 0.04.sup.+++
0.5 mg/kg
Cytochome C
0.54 .+-. 0.04**.sup.x
0.48 .+-. 0.09
1.05 .+-. 0.07.sup.+
0.90 .+-. 0.08.sup.xxx
5 mg/kg
(n = 6)
__________________________________________________________________________
Comparison to: normal*; control.sup.+ ; NAD medication in combination wit
cytochrome C.sup.x
No symbol not statistically significant.
One symbol P < 0.05; two symbols P < 0.01; three symbols P < 0.001
TABLE 9
__________________________________________________________________________
Levels of rabbit cardiac adenyl nucleotides in experimental 10-day TAM
under effect inosine
Left ventricle Right ventricle
mcM/g wet tissue
Energetic
AdT/
mcM/g wet tissue
Energetic
AdT/
GROUP AdT ADP AMP Total
potential
ADP ATP ADP AMP Total
potential
ADP
__________________________________________________________________________
Normal (n = 1)
1 3.23 .+-.
1.49 .+-.
0.71 .+-.
5.36 .+-.
0.73 .+-.
2.23 .+-.
2.75 .+-.
1.47 .+-.
0.74 .+-.
4.89 .+-.
0.68
1.86 .+-.
0.09
0.09
0.13
0.12
0.02 0.18
0.26
0.17
0.03
0.43
0.02 0.18
MYO-
CARDITIS
Control 2 1.51 .+-.
1.27 .+-.
0.69 .+-.
3.48 .+-.
0.62 .+-.
1.26 .+-.
1.33 .+-.
1.24 .+-.
0.73 .+-.
3.29 .+-.
0.59
1.15 .+-.
(n = 7) 0.09
0.13
0.08
0.20
0.02 0.13
0.12
0.16
0.05
0.26
0.01 0.09
Inosine
therapy
80 mg/kg
3 2.66 .+-.
1.50 .+-.
0.99 .+-.
5.14 .+-.
0.66 .+-.
1.82 .+-.
2.25 .+-.
1.74 .+-.
0.63 .+-.
4.82 .+-.
0.65
1.41 .+-.
(n = 6) 0.13
0.10
0.05
0.17
0.01 0.18
0.43
0.21
0.08
0.29
0.01 0.21
160 mg/kg
4 2.99 .+-.
2.36 .+-.
0.98 .+-.
6.20 .+-.
0.65 .+-.
1.25 .+-.
2.23 .+-.
1.85 .+-.
0.96 .+-.
0.96 .+-.
0.63
1.39 .+-.
0.11
0.15
0.16
0.31
0.0 0.12
0.20
0.25
0.06
0.23
0.02 0.30
Least P.sub.1-2
<0.001
-- -- 0.001
0.001 0.001
0.001
-- -- -- 0.01 0.05
significant
P.sub.2-3
<0.001
-- 0.01
0.001
-- 0.05
0.001
-- -- -- 0.01 --
difference
P.sub.2-4
<0.001
0.001
-- 0.001
-- -- 0.01
-- 0.05
0.01
0.001 --
between groups
P.sub.1-3
<-- 0.001
-- 0.02
-- 0.02
-- -- -- -- -- --
(P) P.sub.1-4
<0.01
-- -- -- -- -- -- -- -- -- -- --
P.sub.3-4
<-- 0.001
-- 0.02
-- 0.001
-- -- -- -- -- --
__________________________________________________________________________
TABLE 10
______________________________________
Effect of Refrakterin on left-ventricular tension generated by
PGVM in a 10-day TAM
Statictical
Tension,
Group, subgroup index mH/mm.sup.2
______________________________________
Normal 1 M .+-. m.sub.x
2.39 .+-. 0.34
TAM
Control 2 M .+-. m.sub.x
1.37 .+-. 0.15
Refrakterin 3 M .+-. m.sub.x
2.31 .+-. 0.26
treatment
P.sub.1-2 0.05
P.sub.1-3 --
P.sub.2-3 0.05
______________________________________
TABLE 11
______________________________________
Effects of cardiac glycosides on contraction ability and energy
metabolism of normal myocardial contractile proteins
(.DELTA.H - .DELTA.Q)/
Cardiac glycoside
P .DELTA.G
P/.DELTA.G
.DELTA.H
.DELTA.Q
______________________________________
b-Acetyldigoxin
136.5 155 90.2 95.6 102
10.sup.-6 M
b-Methyldigoxin
10.sup.-8 M
168.8 133.9 189.4 112.6 98.5
10.sup.-6 M
154.6 86.9 142.1 88.2 100
Strophanthin
10.sup.-6 M
168.8 150.5 131.7 117.6 110.2
10.sup.-8 M
149.8 83.0 142.1 90.3 95.6
______________________________________
Note:
given is percentil to Control (no glycosides) in the Table.
TABLE 12
______________________________________
Effects of cardiac glycosides on mechnical and thermodynamic
contraction parameters in TAM
Cardiac glycoside
P .DELTA.H
.DELTA.H - .DELTA.Q/.DELTA.H
______________________________________
Control (10-day TAM)
43.4 52.5 77.9
b-Acetyldigoxin 10.sup.-6 M
75.6 63.0 76.2
Strophanthin K 10.sup.-6 M
75.6 65.7 94.1
______________________________________
Note:
given is percentil to Control (normal) in the Table.
TABLE 13
__________________________________________________________________________
Effect of Refrakterin on contractile protein fiblil/mitochondrial
Ca.sup.2+ transport
in treatment of rabbit TAM (mcmole Ca per g protein
Ca
binding
Statis-
to CPF Ca Mitochrondrial
tical
CPF CPF Ca.sup.2+ uptake
release Ca.sup.2+ uptake
Group index
1 min.
3 min.
15 min.
35 min.
45 min.
load release
5 min.
15 min.
30
__________________________________________________________________________
min.
Normal
1 X .+-. m.sub.x
43.5
834 1298 1426 1414 1345 276 122 196 210
10-day 1.9 50 37 23 19 29 21 11 8 8
15 15 15 15 15 15 15 15 15 15
TAM
Control
2 X .+-. m.sub.x
29.4
563 780 905 908 938 155 171 268 269
0.9 25 39 37 23 35 28 17 22 18
10 7 7 7 7 7 7 9 9 9
Refrakterin
3 X .+-. m.sub.x
40.4
855 1473 1495 1449 1382 226 140 249 235
treatment 2.1 99 142 149 152 167 18 18 41 21
5 6 6 5 6 5 5 5 5 5
P.sub.1-2
0.001
0.01
0.001
0.001
0.001
0.001
0.03
0.01
0.01
0.01
P.sub.1-3
-- -- -- -- -- -- -- -- -- --
P.sub.2-3
0.001
0.01
0.001
0.01 0.01 0.01 -- -- -- --
__________________________________________________________________________
TABLE 14
__________________________________________________________________________
Myocardial CPF Ca.sup.2+ transport in rabbit TAM after oxyfedrin
treatment (mcmole Ca per g protein)
Ca.sup.2+ uptake Ca release
Group 3 min.
5 min. 15 min.
35 min.
45 min.
2 min.
__________________________________________________________________________
Normal M .+-. m
834 .+-. 50
945 .+-. 37
1297 .+-. 27
1426 .+-. 23
1414 .+-. 19
276 .+-. 21
(15) (15) (15) (15) (15) (15)
10-day
TAM
Control M .+-. m
563 .+-. 25
608 .+-. 128
780 .+-. 39
905 .+-. 36
908 .+-. 23
155 .+-. 28
(7) (7) (7) (7) (7) (7)
TREATMENT
P 0.001 0.001 0.001 0.001 0.001 0.01
Oxyfedrin
M .+-. m
1058 .+-. 97
1108 .+-. 74
1319 .+-. 61
1424 .+-. 42
1438 .+-. 44
332 .+-. 36
0.6 mg/kg (5) (5) (5) (6) (6) (6)
P -- -- -- -- -- --
P* 0.001 0.001 0.001 0.001 0.001 0.001
__________________________________________________________________________
P comparisom to normal, P* comparison to Control
TABLE 14a
__________________________________________________________________________
Effects of nonachlazin and oxyfedrin on cardiac AC activity
Activity, pcmole cAMP/mg protein/min.
Test Nonachlazin Oxyfedrine
conditions
Basal
10.sup.-6 M
10.sup.-4 M
10.sup.-6 M
10.sup.-4 M
__________________________________________________________________________
Prior to
142 .+-. 2
-- -- -- --
preincuba-
tion
n = 4
After -- 175 .+-. 3***
162 .+-. 10**
200 .+-. 15***
189 .+-. 9**
preincuba-
tion
n = 4
__________________________________________________________________________
n number of recurrences
Comparison to: basal activity (*) one symbol is P <0.05, two symbols P
<0.01, three symbols P <0.001
TABLE 14b
__________________________________________________________________________
Effects of nonachlazin and oxyfedrin on cardiac PDE activity
Activity, pcmole cAMP/mg protein/min.
Test Nonachlazin Oxyfedrin
conditions
Basal
10.sup.-6 M
10.sup.-4 M
10.sup.-6 M
10.sup.-4 M
__________________________________________________________________________
Prior to
744 .+-. 71
-- -- -- --
preincuba-
tion
n = 4
After -- 526 .+-. 47**
408 .+-. 24***
543 .+-. 59***
402 .+-. 26***
preincuba-
tion
n = 5
__________________________________________________________________________
n number of recurrences
Comparison to: original activity (*) one symbol is P <0.05, two symbols
<0.01, three symbols P <0.001
TABLE 14c
______________________________________
Effects of nonachlazin and oxyfedrin on myocardial cAMP
level in normal rabbit and with TAM
Medicinal cAMP,
Group agent pcmole/mg protein
______________________________________
Control n = 5 1 9.5 .+-. 0.06
Nonachalazin
2 12.5 .+-. 0.07
n = 4
Oxyfedrin 3 14.3 .+-. 0.4*
n = 4
10-day n = 4 4 3.4 .+-. 0.2
TAM Nonachlazin
5 13.4 .+-. 0.3.sup.+++
n = 4
Oxyfedrin 6 12.0 .+-. 0.4.sup.+++o
n = 4
______________________________________
n number of recurrences
*least significant difference between 1-2 and 1-3
.sup.+ least significant difference between 4-5 and 4-6
.sup.o least significant difference between 1-2 and 1-3
One symbol is P <0.05, two symbols P <0.01, three symbols P <0.001
TABLE 15
__________________________________________________________________________
Effects of Refrakterin on levels of adenyl nucleotides and CP in a 10-day
TAM, (.mu.cM/g wet tissue)
Total Phospho-
Group,
Statistic nucleo-
Energetic
rylation
subgroup
value ATP ADP AMP Pn ATP/ADP
tides
potential
index
CP Creatine
__________________________________________________________________________
Normal
1 M .+-. m
3.11
2.1 0.67
2.21
1.5 5.88
0.71 1.48 3.26
15.5
0.18
0.44
0.10
0.19
0.09 0.41
0.014 0.21 0.14
1.9
6 6 6 5 6 6 6 5 6 5
TAM
Control
2 M .+-. m
1.82
1.57
0.84
2.26
1.1 4.24
0.61 2.1 1.69
19.3
0.07
0.07
0.032
0.07
0.08 0.08
0.008 0.37 0.23
2.31
9 9 9 5 9 9 9 5 7 4
Refrakterin
3 M .+-. m
3.13
2.4 0.74
2.13
1.43 6.32
0.58 1.56 3.12
18.22
treatment 0.08
0.14
0.072
0.15
0.07 0.21
0.006 0.17 0.009
0.77
11 11 11 7 11 11 11 7 7 6
P.sub.1-2
0.001
0.01
-- -- 0.01 0.001
0.001 -- 0.001
--
P.sub.1-3
-- -- -- -- -- -- -- -- 0.001
--
P.sub.2-3
0.001
0.001
-- -- 0.05 0.001
0.001 -- -- --
__________________________________________________________________________
TABLE 16
__________________________________________________________________________
Comparison of Refrakterin effectiveness to Ildamen-novodigal
Control
10-day REFRAKTERIN Prior art,
Ingredients TAM MI SK Ildamen + Novodigal
__________________________________________________________________________
Nicotinamide adenine
-- 0.25 0.5 0.5 5 5 -- -- --
dinunucleotide (NAD)
Cytochrome C -- 5 10 10 10 15 -- -- --
Inosine -- 20 80 80 80 250 -- -- --
Cardiac b-Acetyldigoxin
-- 0.05 0.08 -- -- 0.2 0.08
0.15
0.2
glycoside
b-methyldigoxin
-- -- -- 0.008
-- -- -- -- --
Strophanthin K
-- -- -- -- 0.03
-- -- -- --
b-Adrenoceptor
Oxyfedrin 0.3
--
0.3
--
0.3 0.3 2.0
--
0.3 0.6 2.0
stimulant
Nonachlazin* -- 1 -- 1 -- -- -- 3.5
-- -- --
Effect on the
Myocardial
50 109 109.5
108.9
112.6
126 121 155 239
contractile
myofibril
protein system
tension
Effect on
CPF Ca binding
67.6 92.9 90 92.6
Ca transport
CPF Ca uptake
69.8 84.7 102.5 101.7
101 106.9
CPF Ca release
56.0 81.9 110 116.7
Mitochondrial
128 111.9 96.2 58
Ca uptake
Effect on
ATP 58.5 95 100.6
-- 95.5 88.6
52.2
69.1
energy exchange
ADP 74.8 89 114.3
-- 125.4
76.2
86.3
119.8
AMP 125.4
91 110.4
-- 120 60.0
26.3
121.1
ATP/ADP 73.3 82 88.7
-- 100
CP 51.8 98 95.7
-- 96.4 58.3
NADH/NAD 172 125 92.0
-- 105.2
__________________________________________________________________________
Note:
*Nonachlazin-10{b[1,4diazobicyclo(4,3,0)nonanyl-4-propionyl]2-chlorophena
ine dihydrochloride
Claims
1. An improved cardiotropic composition containing a cardiac glycoside and.beta.-adrenergic receptor stimulant wherein the improvement comprises the inclusion of nicotinamide adenine dinucleotide, cytochrome C and inosine, wherein the composition comprises by parts by weight:
2. A cardiotropic composition according to claim 1, wherein the cardiac glycoside is.beta.-acetyldigoxin in an amount of from 0.05 to 0.2 parts by weight.
3. A cardiotropic composition according to claim 1, wherein the cardiac glycoside is.beta.-methyldigoxin in an amount of from 0.05 to 0.15 parts by weight.
4. A cardiotropic composition according to claim 1, wherein the cardiac glycoside is strophanthin K in an amount of from 0.05 to 0.2 parts by weight.
5. A cardiotropic composition according to claim 1, wherein the.beta.-adrenergic receptor stimulant is oxyfedrin in an amount of from 0.3 to 2.0 parts by weight.
6. A cardiotropic composition according to claim 1, wherein the.beta.-adrenergic receptor stimulant is nonachlazin in an amount of from 1 to 3.5 parts by weight.
7. A cardiotropic formulation comprising a cardiac glycoside selected from the group consisting of.beta.-acetyldigoxin,.beta.-methyldigoxin and strophanthin K; a.beta.-adrenergic receptor stimulant selected from the group consisting of oxyfedrin and nonachlazin; nicotinamide adenine dinucleotide; cytochrome C and inosine, wherein the composition comprises by parts by weight;
8. A method for simultaneous restoration of ATP production, transmembrane calcium transport and the cardiomyocyte contraction--relocation system which comprises administering to a patient suffering from disruption of one or more of these mechanisms a dose effective to restore said mechanisms of a composition comprising:
- a cardiac glycoside a.beta.-adrenergic receptor stimulant, nicotinamide adenine dinucleotide, cytochrome C and inosine, wherein the composition comprises by parts by weight:
9. A method according to claim 8, wherein the cardiac glycoside is.beta.-acetyldigoxin in an amount of from 0.05 to 0.2 parts by weight.
10. A method according to claim 8, wherein the cardiac glycoside is.beta.-methyldigoxin in an amount of from 0.05 to 0.15 parts by weight.
11. A method according to claim 8, wherein the cardiac glycoside is strophanthin K in an amount of from 0.05 to 0.2 parts by weight.
12. A method according to claim 8, wherein the.beta.adrenergic receptor stimulant is oxyfedrin in an amount of from 0.3 to 2.0 parts by weight.
13. A method according to claim 8, wherein the.beta.-adrenergic receptor stimulant is nonachalzin in an amount of from 1 to 3.5 parts by weight.
14. A method according to claim 8 wherein the composition used comprises a cardiac glycoside selected from the group consisting of.beta.-acetyldigoxin,.beta.-methyldigoxin and strophanthin K; a.beta.-adrenergic receptor stimulant selected from the group consisting of oxyfederin and nonachlizin; nicotinamide adenine dinucleotide; cytochrome C and inosine, wherein the composition comprises by parts by weight;
- Kostin, V. I. (Dep. Pharmacol., Med. Inst., Kemerovo 650029, USSR). Farmakol. Toksikol. (Moscow), 52(6), 49-52 (Russ) 1989. Karsanov et al. (Repub. Res. Cent. Med. Biophys., Tbilisi, USSR), Izv. Akad. Nauk Gruz. SSR, Ser. Biol., 8(6), 393-9 (Russ) 1982.
Type: Grant
Filed: Jun 28, 1995
Date of Patent: Feb 11, 1997
Inventors: Nikolai V. Karsanov (Tbilisi), Galina V. Sukoian (Rustavi), Zinaida G. Khugashvili (Tbilisi), Dali R. Tatulashvili (Tbilisi), Evgenia V. Selikhova (Tbilisi), Nodar N. Kipshidze (Tbilisi), Eteri I. Guchua (Tbilisi)
Primary Examiner: Phyllis G. Spivack
Law Firm: Ladas & Parry
Application Number: 8/495,298
International Classification: A61K 31135; A61K 3154; A61K 31705; A61K 3816;
